This invention pertains to the field of purification and/or clarification of exopolysaccharides, i.e., polysaccharides produced by microorganisms (referred to herein as polysaccharide-producing bacteria). In particular, this invention relates to methods for the removal of cellular debris and the like which normally accompany fermentation processes utilizing the microorganisms which produce polysaccharides.
Such exopolysaccharides can be precipitated directly from fermentation broths by the addition of an organic solvent in which they are insoluble, e.g., isopropyl alcohol (IPA) and then dried. A solution of the crude or technical-grade polysaccharide in water is turbid due to the presence of cellular debris and insoluble components from the fermentation medium. As used herein, cellular debris includes dead cells and portions thereof present in fermentation broths and precipitated material therefrom in exopolysaccharide production fermentations.
For many end use applications, the particulate contaminating material is undesirable and a more pure product is required. However, procedures to clarify or purify the desired products add complexity and additional cost to the production process.
A typical example of such a polysaccharide is xanthan gum (sometimes referred to herein as xanthan). Thus, for example, when xanthan is used for tertiary oil recovery, the particulate material present in the crude xanthan plugs the pores of the oil-bearing rock formation, thereby reducing oil flow. Also, in cosmetic end uses, wherein xanthan is used as a viscosity control agent, the formulations often require transparency and the xanthan gum must have a high purity.
In order to clarify fermentation broths containing xanthan, chemical, mechanical and enzymatic treatments have been used. Mechanical clarification by either centrifugation or filtration is difficult and time consuming due to the extreme viscosity of the fermentation broths, even though the process can be somewhat improved by warming the xanthan before filtration. See U.S. Pat. No. 4,135,979.
Chemical clarification is carried out by the addition to the fermentation broth of alkali as disclosed in U.S. Pat. Nos. 3,355,447 and 3,964,972 or hypochlorite, as disclosed in U.S. Pat. Nos. 3,516,983, and 3,996,618. However, these chemical treatments also result in deacetylation and degradation of the xanthan polymers.
Several enzymatic methods for clarification of xanthan have been described. See U.S. Pat. Nos. 4,010,071; 4,165,257; GB Patent Application 2,065,689; European Patent Appl. 0,039,962; European Patent Appl. 0,078,621; and U.S. Pat. No. 4,119,491. Each method includes the use of an acidic, neutral or alkaline protease of the type obtained from gram-positive bacteria or fungi. The optimal reaction time, temperature and pH are specific for each enzyme. In at least one case, the enzymatic preparation includes both protease and glucanase activities. See European Patent Application No. 0,039,962.
A variation of the enzymatic method is to add viable and large-sized Trichoderma viride fungal cells to the xanthan broth to consume the xanthan-producing cells. See U.S. Pat. No. 4,094,739. However, this also requires filtration to remove the fungal cells from the viscous fermentation broth.
Generally, it has been thought that gram-negative bacteria would be less likely to serve as a useful source of enzymes for the lysis of other gram-negative microorganisms. This is based on the assumption that the enzyme-secreting bacteria might themselves be damaged by the lytic enzymes that they secrete. As a result, efforts at clarification of cultures of the gram-negative Xanthomonas campestris have thus far focused on the proteolytic enzymes of the gram-positive bacteria, such as, Bacillus subtilis, and eukaryotic fungi.